Soil survey was conducted at four different locations of Guangxi, China to investigate the effect of peanut and soybean inter-cropping in sugarcane cultivation on the soil properties and diazotrophic bacterial diversity. Principal component analysis result shown (PCA) showed difference impact of intercropping and monoculture on the basis of biological and chemical properties of soil. Two-way analysis of variance of soil properties showed a significant interactive effect with locations and cultivation systems. Microbial enumeration results demonstrated that intercropping system enhanced the diazotrophic population. After isolation of diazotrophs, 21 selected bacteria were characterized by siderophore, phosphate, indole acetic acid, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and nitrogenase assay. Among the identified bacteria, isolate MYSP104 showed maximum nitrogenase activity and isolate MYSS78 showed maximum ACC deaminase activity. Partial 16S rRNA gene sequencing results classified these diazotrophs in six phyla (Firmicutes, α, β, Υ-proteobacteria, actinobacteria and bacteroidetes). Diazotrophs such as Bacillus tequilensis, Variovorax paradoxus, Acidovorax facilis, Leucobacter aridicollis, Streptomyces fimicarius and Pseudomonas nitroreducens were reported for the first time from the sugarcane rhizosphere of Guangxi, China. Venn diagram explained that seven bacterial species (Brevibacterium, Burkholderia, Delftia, Leucobacter, Pseudomonas, Sinorhizobium and Variovorax) were recognized with soybean and sugarcane intercropping cultivation systems. This study concluded that intercropping system could enhance the population of N-fixers in soil. Soybean intercropping influenced the soil chemical and biological properties better than peanut. Moreover, the isolated diazotrophs need further characterization and they might be utilized as bio-inoculums for commercial sugarcane production in the future.